Production of foreign proteins in bacterial hosts is now well established. In relatively standard procedures, the gene sequence encoding the desired protein is placed under the control of regulatory sequences indigenous to or compatible with the host and transformed into the host bacterium. In general, there are three major ways in which this can be accomplished: (1) the DNA encoding the desired protein can be fused in reading frame with a bacterial gene already under the control of the bacterial regulatory sequences to obtain a "fusion protein"; (2) the desired coding sequences can be fused in reading frame with an operable leader sequence resulting in a secreted protein; or (3) the desired coding sequence is placed immediately downstream from an ATG start codon which results in "direct" expression to obtain the "mature" protein. In this last instance, the mature protein is not secreted but is found in an intracellular location, often as a refractile or inclusion body.
It is often left unstated, but well recognized by practitioners in the art, that the mature protein formed by the direct expression of ATG-preceded coding sequences frequently bears an N-terminal methionine which is the translation product of the ATG. Depending on the particular recombinant protein, and on the circumstances of its production, more or less of it may be processed in the cell to remove this N-terminal Met residue, but, in general, at least some, and usually a substantial portion of the recombinant protein produced does bear this unwanted foreign residue. Its presence is not completely harmless. When the resulting proteins are used therapeutically, what would ordinarily be an autologous protein to the recipient (for example, human growth hormone (hGH) as administered to humans) now contains a peptide sequence which is unfamiliar to the recipient. The result is predicatable. An immune response may be mounted to the unfamiliar sequence and a therapeutically important peptide now becomes an immunogen.
In addition to foreign proteins, mature native proteins and bacterial proteins from other genera or species are also often incompletely processed. Examples of this phenomenon include E. coli aspartate transcarbamylase (R-chain), E. coli tryptophan synthestase A protein, and E. coli bacteriophage T4 lysozyme. (Fasman, G. O., Ed., CRC Handbook of Biochemistry & Molecular Biology, III:308-313.)
Others have attempted to resolve the N-terminal methionine problem and to produce N-terminal "Met-less" peptides or proteins in various ways. Baxter (U.S. Pat. No. 4,350,764) uses in vitro treatment with the protease trypsin to cleave a precursor protein after protecting alternate cleavage sites. Fusion proteins have also been synthesized where the desired coding sequence is preceded by ATG, thus providing an "internal" methionine in the fusion cleavable by CNBr. Not only does this involve an extra preparation step, but has the more serious defect that the protein or peptide is also cleaved at any methionine residues in the remaining sequence. EPO publication 127,305, published Dec. 5, 1984, to Genentech discloses the production of Met-less hGH by employing a coding sequence which includes the native hGH leader peptide which apparently is workable in certain bacterial hosts to effect secretion of the resulting hGH. Gilbert (U.S. Pat. No. 4,338,397) has employed the penicillinase leader sequence to effect the secretion of presumably N-terminal Met-less .beta.-globin. U.S. Ser. No. 715,653, filed Mar. 25, 1985, assigned to the same assignee and incorporated herein by reference, discloses the use of the leader sequence for bacterial phospholipase A (phoA) to effect secretion of certain, but not all, recombinant peptides.
None of the foregoing approaches provides a universal solution to the problem. Faced with the necessity to produce any particular recombinant protein in N-terminal Met-less form, the practitioner needs to select from a repertoire of possibilities a procedure suitable for the particular peptide to be produced. The method of the invention described below expands this repertoire to provide still another pattern of applicability.